The successful development of an automated cryoEM pipeline for helical specimens in the previous grant period and the subsequent establishment of the National Resource for Automated Molecular Microscopy sets the stage for this current application. One of the critical missing components in automated cryoEM is the means to determine unambiguous initial models for an unknown macromolecular complex and the infrastructure to analyze the degree of conformational heterogeneity in the specimen. The ability to determine the structure of macromolecular complexes in a variety of conformational states is the next big challenge for cryoEM, representing a unique opportunity to address interesting biological questions. Our contribution to this challenge will be to design, implement and test a system for single particle structural analysis that uses experimental, rather than computational, strategies for calculating an initial model, determining the degree of conformational and orientational variability in a specimen, and rejecting imperfect particles during refinement. To achieve this we will develop procedures to determine sets of initial models using both tomographic and random conical data acquisition methods, and procedures for large scale data collection of tilted pairs of images. The data acquisition and analysis procedures will be supported by a web based graphical user interface that will be tightly integrated with a relational database. This work represents a significant extension of the current efforts underway at the National Resource for Automated Molecular Microscopy (NRAMM).